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The induction of permeability in egg-shells of Ascaris suum prior to hatching
THE INDUCTION OF PERMEABILITY IN EGG-SHELLS ASCARIS SUUM PRIOR TO HATCHING
OF
A. J. CLARKE and R. N. PERRY AFRC Institute for Arable
Crops
Research, Nematology Department, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, U.K. (Received 6 August 1987; accepted 11 May 1988)
A. J. and PERRY R. N. 1988. The induction of permeability in egg-shells of Ascaris SUUM International Journal for Parasitology 18: 987-990. X-ray micro-analysis was used to study changes in the Na+ content of egg-shells of Ascaris suum. Egg-shells isolated from untreated eggs contained little Na+ but egg-shells recovered from eggs hatched in vitro contained substantial amounts of
fibStraCt-CLARKE
prior to hatching.
Na+. Similarlv, egg-shells isolated from eggs treated with NaHCO, contained much Na+, as did shells obtained from h&-treated eggs exposed-to the same medium. Egg-shells treated with NaHCO, also contained Na+ not readilv removed bv washina. However, free shells treated successively with NaHCO, and trehafose retained little ha+ indicating that the Na+ is not ionically bound. The Na+ may form a complex with the ascaroside membrane, and the results are discussed in relation to the properties of the membrane. A hatching mechanism is outlined. INDEX
KEY WORDS:
Nematode;
Ascaris mum; hatching; egg-shell; sodium; permeability
A. sum
egg-shells are glycosides of the rare sugar, (3, 6-dideoxy-r.-arabinohexose) and long chain secondary mono1 and diol alcohols. The major ascaroside (Tarr & Fairbairn. 1973) present in the egg-shell (about 70% of the total ascarosides) is a diol ascaroside with an acetylated aglycone. The composition of the ascarosides of A. mum eggs is given by Jezyk & Fairbairn (1967) Tarr & Fairbaim (1973) and Tarr & Schnoes (1973). Although both the conditions necessary for hatching and the basic composition of the membrane substrate are known, the mechanism whereby the permeability of the ascaroside membrane is changed is unknown. We used X-ray microanalytical methods to investigate the possibility that Na+ ions are involved in the alteration in permeability of this membrane.
INTRODUCTION Ascaris lumbricoides and A. mum may survive in the soil for several years in a dormant state but, after ingestion by a host, the infective egg hatches rapidly in response to the stimulus provided by the alimentary tract. Hatching can also be effected in vitro at 38°C by bubbling CO, through a suspension of eggs in a bicarbonate medium (Rogers, 1958; Fairbairn, 196 1; Perry & Clarke, 1982). The presence of a reducing agent enhances hatching but is not essential. The egg-shell which protects the juvenile throughout dormancy has four layers, the outermost of which is the uterine layer, consisting of glycoprotein, and is followed successively by a thin vitelline layer, a chitinous layer and an innermost lipid layer, termed the ascaroside membrane (Wharton, 1986). An early stage in the hatching process is the change in permeability of the previously largely impermeable ascaroside membrane. The change is detected by the release of trehalose from the egg into the surrounding medium (Fairbairn, 1961) with a concomitant increase in water content of the unhatched juvenile (Clarke & Perry, 1980). Enzymes also pass through the newly permeable layer and attack the outer eggshell layers. Although the permeability of the ascaroside layer is altered, it remains intact and no chemical change can be detected (Barrett, 1976). Chemically, the layer is a lipoprotein, the lipid component of which consists almost entirely of ascarosides (Fairbairn & Passey, 1955). Ultrastructural studies (Lysek, Malinsky & Janish, 1985; Sromova, 1986) show the laminar nature of the membrane. The ascarosides of THE
eggs
ascarylose
of
MATERIALS AND METHODS A. mum eggs were isolated, embryonated and prepared for use as described by Fairbairn (1970). Fairbairn’s hatching medium contained about 0.07 M-NaCl, 0.03 M-NaHSO, and 0.03 M-NaHCG,, with a pH of about 6.8. Bicarbonate solutions were gassed with N2-CO2 (95%5%) for 15 min before use. The viability of the eggs was established by hatching tests in Fairbairn’s medium at 38°C carried out as described previously (Clarke &Perry, 1980); hatches of 7595% were obtained. Batches of eggs were transferred to glass distilled water (GDW) and rinsed several times in fresh GDW before the start of each set of experiments. Techniques for treating whole and ruptured eggs and for preparing egg-shells for X-ray microanalysis were similar to those previously described for G. rostochiensis by Clarke & Perry (1985). All analyses were carried out on empty egg shells. 987
988
A. J.
CLARKE
and R. N.
For experiments where treatments were on empty eggshells, samples of egg suspensions were added to microscope slides, covered with a glass cover shp and eggs ruptured by gentle pressure on the cover slip. Juveniles were discarded and the egg-shells transferred to 6-mm glass cylinders with 45pm mesh nylon netting tixed over the lower end (Forrest & Perry. 1980). Each cylinder was placed in a 9ml capacity capped vial cont~ning the experimental solution and kept at 3 “C. Solutions were changed by transferring the cylinder to a vial of fresh solution. For experiments where treatments were on whole eggs containing juveniles, the procedure was as above except that eggs were ruptured at the end of the treatment, after rinsing with GDW, and the egg-shells transferred to a cylinder-sieve for further treatment or to foil for ashing. For experiments with heat-treated egg-shells, whole eggs were placed in boiling distilled water (100°C) for 5 min before being transferred to the experimental solutions. When all treatments had been completed, egg-shells were rinsed in GDW and pipetted onto small pieces of aluminium foil using a 25-~1 Drummond Microcap pipette. After excess GDW had been removed, the egg-shells were ashed in a Plasmaprep 100 low temperature ashing unit (Nanotech Thin Films Ltd) for 15 min at 100 W radio frequency power (13.56 MHz) and a pressure of 133 Pa of oxygen at a temperature not exceeding 50°C. The foil pieces were then attached to carbon stubs with a carbon based cement. Eggshells were examined in a scanning electron microscope (Cambridge Instruments S250) at 20 kV accelerating voltage and X2000 magnificatiotl. Elements in the shell (given as mole % element per unit area) were determined with a Link System 290 energy dispersive X-ray microanalyser (100 s live time). Analyses were performed in the raster mode, and quantitative data were obtained using a Link Svstems ZAF4 P/B (Peak to Background ratio1 program. The egg-shell has a thickness which G less than the excitation volume generated by the electron beam and therefore a large A13+ peak was generated from the foil supporting the eggs. Since this A13+ peak and its associated background would make it difficult to measure Na+ with any accuracy, they were stripped from the spectrum prior to analysis. The beam current was set by adjusting the absorbed current to 1.2 X 1 O-I” A measured on a pure cobalt standard near the specimens before each analysis. Results are given as mean values for analyses of lo-20 egg-shells per treatment. RESULTS
The X-ray spectrum of the ash of egg-shells treated with GDW only (Table 1, treatment A) showed that the ash contained traces of various metals, none of which was present in an outstanding amount. The Na+ content of 0.28 Z!I0.17% was taken as the reference standard for this element. Table 1 lists various treatments of empty egg-shells and whole eggs, and the results of subsequent Naf analyses of the ashed shells. Egg-shells from A. suum eggs hatched in Fairbairn’s hatching medium (treatment B) retained a significant (P< 0.01) amount of additional Na+, as did egg-shells obtained from whole eggs treated with 0.1 MNaHCO, only (treatment C). Shells from heat-treated whole eggs subjected to immersion in 0.1 M-NaHCG,I (treatment D) also contained appreciably more Na+ than the standard. as did free egg-she& treated with 0.03 M-NaHCG, (treatment E). No Na+ was present in egg-shells treated with a solution of 0.03 M-
PERRY
NaHCO, in 0.2 M-trehalose (treatment F) and after the successive treatment of egg-shells with 0.03 hf*1NaHCO, and 0.2 M-trehatose (treatment G), only relatively small amounts of Na+ remained in the shells. DISCUSSION
Table 1 shows that after treatments B, C, R or E, A. SUUM egg-shells retained Na+ that was not readily removed by washing. We suggest that the Na+ may be bound at specific sites although we cannot exclude the possibility that the ion is trapped adventitiously. Fairbairn & Passey (1955) found that the ascaroside layer was not permeable to Na+ ions (as NaCl or NaOH). The egg-shell is, however, permeable to water (Clarke & Perry, 1980) and to CO, as is shown by the escape of respiratory CO, and the incorporation within the egg of rJC02 from the surrounding medium (Passey & Fairbairn, 1957). We suggest that free CO,, which is only slowly hydrated (Asada, 1982) penetrates the membrane to occupy interstitial sites present among the ascaroside head groups. At the sites, the CO, molecule is hydrated, and changes from a linear, relaiively non-polar molecule into the larger disc-shaped carbonic acid and its ions (Cotton, Witkinson & Gaus, 1987); at about pH 7 the predominant species is the HCO,- ion. Carbonic acid and its ions are, it is suggested, incompatible, because of size and change, with the environment in which they are formed. A mutual repulsion of ascaroside and HCO,- provides the driving force for a structural change in the membrane, which at about 38’ is already on the threshold of thermally-induced changes in permeability (Barrett, 1976). As a result a new phase is formed with sufficiently large pores to permit the passage of trehalose and enzymes; possibly the new phase is stabilized in the presence of NaHCO, by the formation of a NaHCO,-ascaroside complex. There is evidence that the egg-shell can accommodate inclusion compounds, for isolated ascarosides as well as the ascaroside membrane itself give a blue colour when treated with iodine (Tarr Cyr.Fairbairn, 1973; Barrett, 1976) and egg-shells treated with H,S acquire a blackish tinge and retain the gas despite extensive washing (Hurley & Sommerville, 1982). Analagous host-guest relationships, e.g. of the cycle-dextrins (Saenger, 1980) have been the subjects of detailed studies. Inhibition of A. mum hatching by iodine and its reversal by treatment with HZ8 (Hurley & Sommervi~ie, 1982) or thiosulphate (Wattal, Malla, Khan & Agarwal, 1985) may be associated with the occupation of the CO, binding sites by iodine, although Hurley & Sommerville (1982) advance other possible explanations. The Na” retained by A. suum egg-shells in our experiments is not ionically bound for it was removed when the egg shells were treated with 0.2 M-trehajose. Trehalose and other sugars can complex mono- and divalent cations (Poonia & Bajaj. 1979) and it is assumed binding
that sites
trehalose for the
competes Na+ ions.
with Other
the egg-shell evidence of
of A. sum
Egg permeability ‘GABLE
~-THE
Na+coNrsN’r
(MOLE
“6)
f
STANDARD
ERROR
S.E.M.
OF TREATMENTSIS FROM Whole eggs Treatment A B
Duration 2X24h 18h
Egg-opening medium?
THE SEQUENCE
TO RIGHT
Opened egg-shell Treatment
Duration
Analyses (after ashing) Mole %Na+ i S.E.M.
GDW GDW
rinse rinse
0.28 + 0.17 20.19 4 2.61
GDW
GDW
rinse
17.01 f 2.73
GDW GDW
GDW *(a) O.O3M-NaHCO~ (b) GDW *(a) 0.03 M-NaHCO, plus 0.2 M-trehdose (b) GDW *(a) 0.03 ~-NtiCo,
rinse 24h rinse 24 h
19.34 Ik 2.07
rinse 24 h rinse 24 h rinse
0
E
rinse 24 h rinse 5 min 24 h rinse rinse
F
GDW
rinse
GDW
G
GDW
rinse
GDW
D
ASHED EGG-SHELLS OF Ascarissuum.
LEFT
GDW Fairbairn’s medium
GDW Fairbairn’s medium *(a) GDW (b) 0.1 M-NaHCO, (c) GDW *(a) boiling GDW @) 0.1 M-NaHCO, (c) GDW GDW
C
OF
989
@) GDW (c) 0.2 M-trehalose (d) GDW
10.55 i 2.57
1.52 k 0.60
‘GDW rinse’ denotes three successive washes with glass distilled water. *Denotes consecutive treatments. tFor all the experiments except B the eggs were opened in the medium by mechanical rupture of the shell; for experiment the shells were obtained after the juveniles had been stimulated to hatch in Fairbairn’s medium.
cation binding is given by Holah (unpublished Ph.D. thesis, Trent Polytechnic 1986), who reported that the ascaroside layer, isolated from A. mum eggs submitted to prolonged immersion in Ca(OH), solution, contained bound Ca?+. There are also experiments which may connect NaHCO, binding more directly to a permeability change in the egg-shell. Barrett (1976) used the fluorescent molecular probe, 1 -anilino-S-naphthalenesulpho~c acid (ANS), as an indicator of phase change in ascaroside membranes subjected to various treatments. In only one set of experiments was any change in the probe’s fluorescence characteristics detected. The experiments involved eggs from which the protein coat was removed, i.e. decoated by treatment with 0.5 M-NaOH, after which the eggs were washed with water. The enhanced fluorescence was attributed to the effect of traces of protein left on the surface of the egg-shell, but an alternative interpretation of the results is that in the preparation of the decoated eggs the egg membranes were exposed to similar conditions of mild alkali and dissolved CO? to those necessary for hatching, so that the membranes were in a permeable state when treated with ANS. The molar ratios of CO,, H,CO,, HCO,and C0,2- at various pHs can be calculated from eyns (1) and (2) (Asada, 1982), where the first and second dissociation constants are pK,, = 6.35 and pK,, = 10.32, respectively.
pH=PKa4 +log[H,CO,
+ CO,]
B
(1)
[co,2-] pH = pK,, + log ~ IHCO,-1
(2)
The molar ratio of H&O, is 0.26% of the concentration of H&O, + CO,. In keeping with our hypothesis, the pH range for active hatching corresponds with the range over which free CO, and HCO,- co-exist in solution. The enzyme, carbonic anhydrase, which catalyses the reaction CO, + Hz0 * H’ + HCO,-, might be involved in hatching (Hurley & Sommerville, 1982), but there is as yet no experimental evidence of its participation. We obtained evidence of Na+ retention by A. swm egg-shells after their treatment with the hatching medium, and we have outlined a hatching mechanism in which NaHCO, may participate. The mechanism may be common to other ascarid species and, perhaps in view of similarities (Rogers, 1960; Hurley & Sommerville, 1982), may aiso be relevant to the process of exsheathment.
990
A. J. CLARKE a nd R. N. PERRY
REFERENCES ASADA K. 1982. Biological carboxylations. In: Organic and Bio-organic Chemistry of Carbon Dioxide (Edited by INOUE S. & YAMAZAKI N.), pp. 185-2 10. Halstead Press, New York. BARRETTJ. 1976. Studies on the induction of permeability in Ascaris lumbricoides eggs. Parasitology 73: 109-121. CLARKE A. J. & PERRY R. N. 1980. Egg-shell permeability and hatching of Ascarissuum. Parasitolom 80: 447-456. CLARKE A. J. & PERRY R. N. 1985. Egg-shell calcium and the hatching of Globodera rostorhiensis. International Journaf for Parusitology 1.5: S 11-S 16. COTTON E. A.. WILK~SON G. & GAL’S P. L. 1987. Basic Inorganic Chemists, 2nd Edn. John Wiley & Sons, New York. FAIRBAIRND. &PASSE~ B. 1. 1955.Thelipidcomponentsin the vitelline membrane of A.scaris lumbrico;des eggs. Canadian Journal of Biochemistvv and Phvsiolonv -. 33: 130-134. FAIRBAHW D. 196 1. The in vifro hatching of Ascaris Iumbricoides eggs. Cunadiun Journal ofZoology 39: L53-l 62. FAIRBAIRV D. 1970. Physiological hatching of AX&$ lunzbricoides eggs. In: Experiments and Techniques in Parasitology (Edited by MACINNK A. J. 6t VOGE M.). pp. 21-23. W. H. Freeman, San Francisco. FORKEST J. M. S. & PERRY R.N. 1980. Hatching of Globodera pallida after brief exposures to potato-root diffusate. IVenzato~o~~~iz 26: 130-l 32. HURLEY L.C. & SO~~~R~~~~~ R.J. 1982. Reversible inhibition of hatching of infective eggs of Ascrrric suutn (Nematoda). lntemutional Journal for Parasitology 12: 463-465. JEZYK P. F. & FAIRBAIRND. 1967. Ascarosides and ascaroside esters in Ascaris lumbricoides (Nematoda). Comparative Biochemistryand Physiology 23: 691-705. L+SEK H.. MA&SK+ J. & JANISH R. 1985.Ultrastructureof
eggs of Ascaris iumbricoides Linnaeus 1758-i. Eggshells. Folia Parasitologica 32: 381-384. PASSEY R. F. & FAIRBAIRND. 1957. The conversion of fat to carbohydrate during embryonation of Ascaris lumbricoides ” eggs. Canadian Journal of Biochemistry and Ph~~olo~v 35: 51 l-525. PERR; R. N:& CLARKE A. J. 1982. Hatching mechanism of nematodes. In: Trends and Perspectives in Parasitology, Vol. 2 (Edited by CROMPTOND. W. T. &NEWTON B. A.), pp. 63-77. Cambridge University Press, Cambridge. PO~NIA N. S. & BAJAJ A. V. 1979. Co-ordination of alkali and alkaline earth cations. Chemical Reviews 5: 389-446. RICERS W. P. 1958. Physiology of the hatching of eggs of Ascuris ~zinzbr~~o~des.Nature (London) \ , 18 1: 14 1O1411. ROGERS W. P. 1960. The physiology of infective processes of nematode parasites; the stimulus from the animal host. Proceedings of the RoyalSociety Series B 152: 367-386. SAENCER W. 1980. Cyclodextrin inclusion compounds in research and industry. Angewandte Chemie (International Ed~i~on~ 19: 344-362. SROMOVAD. 1986. The formation of ascarosid layer of the egg-shell in Ascuris lumbricoides. Folk Parasitotogica 33: 169-171. TARR G. E. & FAIRBAIRN D. 1973. Ascarosides of the ovaries and eggs of Ascaris htmbricoides (Nematoda). Lipi& 8: 7-l 6. TARR G. E. & SCHNOES H. K. 1973. Structures of ascaroside aglycones. Archives of B~oche~zist~~and Biophysic.~ 158: 288-296. WATTAL C., MALLA N., KHAX I. A. & AGARWAL S. C. 1985. Reversible inhibition of development and hatching of infective eggs of Ascuris lumbricoides var. hominis. Journal of Parasitology 7 1: 5 18. WHARTON D. A. 1986. A Functional BiologyofNematodes. Croom Helm. London.
OF
A. J. CLARKE and R. N. PERRY AFRC Institute for Arable
Crops
Research, Nematology Department, Rothamsted Experimental Station, Harpenden, Hertfordshire AL5 2JQ, U.K. (Received 6 August 1987; accepted 11 May 1988)
A. J. and PERRY R. N. 1988. The induction of permeability in egg-shells of Ascaris SUUM International Journal for Parasitology 18: 987-990. X-ray micro-analysis was used to study changes in the Na+ content of egg-shells of Ascaris suum. Egg-shells isolated from untreated eggs contained little Na+ but egg-shells recovered from eggs hatched in vitro contained substantial amounts of
fibStraCt-CLARKE
prior to hatching.
Na+. Similarlv, egg-shells isolated from eggs treated with NaHCO, contained much Na+, as did shells obtained from h&-treated eggs exposed-to the same medium. Egg-shells treated with NaHCO, also contained Na+ not readilv removed bv washina. However, free shells treated successively with NaHCO, and trehafose retained little ha+ indicating that the Na+ is not ionically bound. The Na+ may form a complex with the ascaroside membrane, and the results are discussed in relation to the properties of the membrane. A hatching mechanism is outlined. INDEX
KEY WORDS:
Nematode;
Ascaris mum; hatching; egg-shell; sodium; permeability
A. sum
egg-shells are glycosides of the rare sugar, (3, 6-dideoxy-r.-arabinohexose) and long chain secondary mono1 and diol alcohols. The major ascaroside (Tarr & Fairbairn. 1973) present in the egg-shell (about 70% of the total ascarosides) is a diol ascaroside with an acetylated aglycone. The composition of the ascarosides of A. mum eggs is given by Jezyk & Fairbairn (1967) Tarr & Fairbaim (1973) and Tarr & Schnoes (1973). Although both the conditions necessary for hatching and the basic composition of the membrane substrate are known, the mechanism whereby the permeability of the ascaroside membrane is changed is unknown. We used X-ray microanalytical methods to investigate the possibility that Na+ ions are involved in the alteration in permeability of this membrane.
INTRODUCTION Ascaris lumbricoides and A. mum may survive in the soil for several years in a dormant state but, after ingestion by a host, the infective egg hatches rapidly in response to the stimulus provided by the alimentary tract. Hatching can also be effected in vitro at 38°C by bubbling CO, through a suspension of eggs in a bicarbonate medium (Rogers, 1958; Fairbairn, 196 1; Perry & Clarke, 1982). The presence of a reducing agent enhances hatching but is not essential. The egg-shell which protects the juvenile throughout dormancy has four layers, the outermost of which is the uterine layer, consisting of glycoprotein, and is followed successively by a thin vitelline layer, a chitinous layer and an innermost lipid layer, termed the ascaroside membrane (Wharton, 1986). An early stage in the hatching process is the change in permeability of the previously largely impermeable ascaroside membrane. The change is detected by the release of trehalose from the egg into the surrounding medium (Fairbairn, 1961) with a concomitant increase in water content of the unhatched juvenile (Clarke & Perry, 1980). Enzymes also pass through the newly permeable layer and attack the outer eggshell layers. Although the permeability of the ascaroside layer is altered, it remains intact and no chemical change can be detected (Barrett, 1976). Chemically, the layer is a lipoprotein, the lipid component of which consists almost entirely of ascarosides (Fairbairn & Passey, 1955). Ultrastructural studies (Lysek, Malinsky & Janish, 1985; Sromova, 1986) show the laminar nature of the membrane. The ascarosides of THE
eggs
ascarylose
of
MATERIALS AND METHODS A. mum eggs were isolated, embryonated and prepared for use as described by Fairbairn (1970). Fairbairn’s hatching medium contained about 0.07 M-NaCl, 0.03 M-NaHSO, and 0.03 M-NaHCG,, with a pH of about 6.8. Bicarbonate solutions were gassed with N2-CO2 (95%5%) for 15 min before use. The viability of the eggs was established by hatching tests in Fairbairn’s medium at 38°C carried out as described previously (Clarke &Perry, 1980); hatches of 7595% were obtained. Batches of eggs were transferred to glass distilled water (GDW) and rinsed several times in fresh GDW before the start of each set of experiments. Techniques for treating whole and ruptured eggs and for preparing egg-shells for X-ray microanalysis were similar to those previously described for G. rostochiensis by Clarke & Perry (1985). All analyses were carried out on empty egg shells. 987
988
A. J.
CLARKE
and R. N.
For experiments where treatments were on empty eggshells, samples of egg suspensions were added to microscope slides, covered with a glass cover shp and eggs ruptured by gentle pressure on the cover slip. Juveniles were discarded and the egg-shells transferred to 6-mm glass cylinders with 45pm mesh nylon netting tixed over the lower end (Forrest & Perry. 1980). Each cylinder was placed in a 9ml capacity capped vial cont~ning the experimental solution and kept at 3 “C. Solutions were changed by transferring the cylinder to a vial of fresh solution. For experiments where treatments were on whole eggs containing juveniles, the procedure was as above except that eggs were ruptured at the end of the treatment, after rinsing with GDW, and the egg-shells transferred to a cylinder-sieve for further treatment or to foil for ashing. For experiments with heat-treated egg-shells, whole eggs were placed in boiling distilled water (100°C) for 5 min before being transferred to the experimental solutions. When all treatments had been completed, egg-shells were rinsed in GDW and pipetted onto small pieces of aluminium foil using a 25-~1 Drummond Microcap pipette. After excess GDW had been removed, the egg-shells were ashed in a Plasmaprep 100 low temperature ashing unit (Nanotech Thin Films Ltd) for 15 min at 100 W radio frequency power (13.56 MHz) and a pressure of 133 Pa of oxygen at a temperature not exceeding 50°C. The foil pieces were then attached to carbon stubs with a carbon based cement. Eggshells were examined in a scanning electron microscope (Cambridge Instruments S250) at 20 kV accelerating voltage and X2000 magnificatiotl. Elements in the shell (given as mole % element per unit area) were determined with a Link System 290 energy dispersive X-ray microanalyser (100 s live time). Analyses were performed in the raster mode, and quantitative data were obtained using a Link Svstems ZAF4 P/B (Peak to Background ratio1 program. The egg-shell has a thickness which G less than the excitation volume generated by the electron beam and therefore a large A13+ peak was generated from the foil supporting the eggs. Since this A13+ peak and its associated background would make it difficult to measure Na+ with any accuracy, they were stripped from the spectrum prior to analysis. The beam current was set by adjusting the absorbed current to 1.2 X 1 O-I” A measured on a pure cobalt standard near the specimens before each analysis. Results are given as mean values for analyses of lo-20 egg-shells per treatment. RESULTS
The X-ray spectrum of the ash of egg-shells treated with GDW only (Table 1, treatment A) showed that the ash contained traces of various metals, none of which was present in an outstanding amount. The Na+ content of 0.28 Z!I0.17% was taken as the reference standard for this element. Table 1 lists various treatments of empty egg-shells and whole eggs, and the results of subsequent Naf analyses of the ashed shells. Egg-shells from A. suum eggs hatched in Fairbairn’s hatching medium (treatment B) retained a significant (P< 0.01) amount of additional Na+, as did egg-shells obtained from whole eggs treated with 0.1 MNaHCO, only (treatment C). Shells from heat-treated whole eggs subjected to immersion in 0.1 M-NaHCG,I (treatment D) also contained appreciably more Na+ than the standard. as did free egg-she& treated with 0.03 M-NaHCG, (treatment E). No Na+ was present in egg-shells treated with a solution of 0.03 M-
PERRY
NaHCO, in 0.2 M-trehalose (treatment F) and after the successive treatment of egg-shells with 0.03 hf*1NaHCO, and 0.2 M-trehatose (treatment G), only relatively small amounts of Na+ remained in the shells. DISCUSSION
Table 1 shows that after treatments B, C, R or E, A. SUUM egg-shells retained Na+ that was not readily removed by washing. We suggest that the Na+ may be bound at specific sites although we cannot exclude the possibility that the ion is trapped adventitiously. Fairbairn & Passey (1955) found that the ascaroside layer was not permeable to Na+ ions (as NaCl or NaOH). The egg-shell is, however, permeable to water (Clarke & Perry, 1980) and to CO, as is shown by the escape of respiratory CO, and the incorporation within the egg of rJC02 from the surrounding medium (Passey & Fairbairn, 1957). We suggest that free CO,, which is only slowly hydrated (Asada, 1982) penetrates the membrane to occupy interstitial sites present among the ascaroside head groups. At the sites, the CO, molecule is hydrated, and changes from a linear, relaiively non-polar molecule into the larger disc-shaped carbonic acid and its ions (Cotton, Witkinson & Gaus, 1987); at about pH 7 the predominant species is the HCO,- ion. Carbonic acid and its ions are, it is suggested, incompatible, because of size and change, with the environment in which they are formed. A mutual repulsion of ascaroside and HCO,- provides the driving force for a structural change in the membrane, which at about 38’ is already on the threshold of thermally-induced changes in permeability (Barrett, 1976). As a result a new phase is formed with sufficiently large pores to permit the passage of trehalose and enzymes; possibly the new phase is stabilized in the presence of NaHCO, by the formation of a NaHCO,-ascaroside complex. There is evidence that the egg-shell can accommodate inclusion compounds, for isolated ascarosides as well as the ascaroside membrane itself give a blue colour when treated with iodine (Tarr Cyr.Fairbairn, 1973; Barrett, 1976) and egg-shells treated with H,S acquire a blackish tinge and retain the gas despite extensive washing (Hurley & Sommerville, 1982). Analagous host-guest relationships, e.g. of the cycle-dextrins (Saenger, 1980) have been the subjects of detailed studies. Inhibition of A. mum hatching by iodine and its reversal by treatment with HZ8 (Hurley & Sommervi~ie, 1982) or thiosulphate (Wattal, Malla, Khan & Agarwal, 1985) may be associated with the occupation of the CO, binding sites by iodine, although Hurley & Sommerville (1982) advance other possible explanations. The Na” retained by A. suum egg-shells in our experiments is not ionically bound for it was removed when the egg shells were treated with 0.2 M-trehajose. Trehalose and other sugars can complex mono- and divalent cations (Poonia & Bajaj. 1979) and it is assumed binding
that sites
trehalose for the
competes Na+ ions.
with Other
the egg-shell evidence of
of A. sum
Egg permeability ‘GABLE
~-THE
Na+coNrsN’r
(MOLE
“6)
f
STANDARD
ERROR
S.E.M.
OF TREATMENTSIS FROM Whole eggs Treatment A B
Duration 2X24h 18h
Egg-opening medium?
THE SEQUENCE
TO RIGHT
Opened egg-shell Treatment
Duration
Analyses (after ashing) Mole %Na+ i S.E.M.
GDW GDW
rinse rinse
0.28 + 0.17 20.19 4 2.61
GDW
GDW
rinse
17.01 f 2.73
GDW GDW
GDW *(a) O.O3M-NaHCO~ (b) GDW *(a) 0.03 M-NaHCO, plus 0.2 M-trehdose (b) GDW *(a) 0.03 ~-NtiCo,
rinse 24h rinse 24 h
19.34 Ik 2.07
rinse 24 h rinse 24 h rinse
0
E
rinse 24 h rinse 5 min 24 h rinse rinse
F
GDW
rinse
GDW
G
GDW
rinse
GDW
D
ASHED EGG-SHELLS OF Ascarissuum.
LEFT
GDW Fairbairn’s medium
GDW Fairbairn’s medium *(a) GDW (b) 0.1 M-NaHCO, (c) GDW *(a) boiling GDW @) 0.1 M-NaHCO, (c) GDW GDW
C
OF
989
@) GDW (c) 0.2 M-trehalose (d) GDW
10.55 i 2.57
1.52 k 0.60
‘GDW rinse’ denotes three successive washes with glass distilled water. *Denotes consecutive treatments. tFor all the experiments except B the eggs were opened in the medium by mechanical rupture of the shell; for experiment the shells were obtained after the juveniles had been stimulated to hatch in Fairbairn’s medium.
cation binding is given by Holah (unpublished Ph.D. thesis, Trent Polytechnic 1986), who reported that the ascaroside layer, isolated from A. mum eggs submitted to prolonged immersion in Ca(OH), solution, contained bound Ca?+. There are also experiments which may connect NaHCO, binding more directly to a permeability change in the egg-shell. Barrett (1976) used the fluorescent molecular probe, 1 -anilino-S-naphthalenesulpho~c acid (ANS), as an indicator of phase change in ascaroside membranes subjected to various treatments. In only one set of experiments was any change in the probe’s fluorescence characteristics detected. The experiments involved eggs from which the protein coat was removed, i.e. decoated by treatment with 0.5 M-NaOH, after which the eggs were washed with water. The enhanced fluorescence was attributed to the effect of traces of protein left on the surface of the egg-shell, but an alternative interpretation of the results is that in the preparation of the decoated eggs the egg membranes were exposed to similar conditions of mild alkali and dissolved CO? to those necessary for hatching, so that the membranes were in a permeable state when treated with ANS. The molar ratios of CO,, H,CO,, HCO,and C0,2- at various pHs can be calculated from eyns (1) and (2) (Asada, 1982), where the first and second dissociation constants are pK,, = 6.35 and pK,, = 10.32, respectively.
pH=PKa4 +log[H,CO,
+ CO,]
B
(1)
[co,2-] pH = pK,, + log ~ IHCO,-1
(2)
The molar ratio of H&O, is 0.26% of the concentration of H&O, + CO,. In keeping with our hypothesis, the pH range for active hatching corresponds with the range over which free CO, and HCO,- co-exist in solution. The enzyme, carbonic anhydrase, which catalyses the reaction CO, + Hz0 * H’ + HCO,-, might be involved in hatching (Hurley & Sommerville, 1982), but there is as yet no experimental evidence of its participation. We obtained evidence of Na+ retention by A. swm egg-shells after their treatment with the hatching medium, and we have outlined a hatching mechanism in which NaHCO, may participate. The mechanism may be common to other ascarid species and, perhaps in view of similarities (Rogers, 1960; Hurley & Sommerville, 1982), may aiso be relevant to the process of exsheathment.
990
A. J. CLARKE a nd R. N. PERRY
REFERENCES ASADA K. 1982. Biological carboxylations. In: Organic and Bio-organic Chemistry of Carbon Dioxide (Edited by INOUE S. & YAMAZAKI N.), pp. 185-2 10. Halstead Press, New York. BARRETTJ. 1976. Studies on the induction of permeability in Ascaris lumbricoides eggs. Parasitology 73: 109-121. CLARKE A. J. & PERRY R. N. 1980. Egg-shell permeability and hatching of Ascarissuum. Parasitolom 80: 447-456. CLARKE A. J. & PERRY R. N. 1985. Egg-shell calcium and the hatching of Globodera rostorhiensis. International Journaf for Parusitology 1.5: S 11-S 16. COTTON E. A.. WILK~SON G. & GAL’S P. L. 1987. Basic Inorganic Chemists, 2nd Edn. John Wiley & Sons, New York. FAIRBAIRND. &PASSE~ B. 1. 1955.Thelipidcomponentsin the vitelline membrane of A.scaris lumbrico;des eggs. Canadian Journal of Biochemistvv and Phvsiolonv -. 33: 130-134. FAIRBAHW D. 196 1. The in vifro hatching of Ascaris Iumbricoides eggs. Cunadiun Journal ofZoology 39: L53-l 62. FAIRBAIRV D. 1970. Physiological hatching of AX&$ lunzbricoides eggs. In: Experiments and Techniques in Parasitology (Edited by MACINNK A. J. 6t VOGE M.). pp. 21-23. W. H. Freeman, San Francisco. FORKEST J. M. S. & PERRY R.N. 1980. Hatching of Globodera pallida after brief exposures to potato-root diffusate. IVenzato~o~~~iz 26: 130-l 32. HURLEY L.C. & SO~~~R~~~~~ R.J. 1982. Reversible inhibition of hatching of infective eggs of Ascrrric suutn (Nematoda). lntemutional Journal for Parasitology 12: 463-465. JEZYK P. F. & FAIRBAIRND. 1967. Ascarosides and ascaroside esters in Ascaris lumbricoides (Nematoda). Comparative Biochemistryand Physiology 23: 691-705. L+SEK H.. MA&SK+ J. & JANISH R. 1985.Ultrastructureof
eggs of Ascaris iumbricoides Linnaeus 1758-i. Eggshells. Folia Parasitologica 32: 381-384. PASSEY R. F. & FAIRBAIRND. 1957. The conversion of fat to carbohydrate during embryonation of Ascaris lumbricoides ” eggs. Canadian Journal of Biochemistry and Ph~~olo~v 35: 51 l-525. PERR; R. N:& CLARKE A. J. 1982. Hatching mechanism of nematodes. In: Trends and Perspectives in Parasitology, Vol. 2 (Edited by CROMPTOND. W. T. &NEWTON B. A.), pp. 63-77. Cambridge University Press, Cambridge. PO~NIA N. S. & BAJAJ A. V. 1979. Co-ordination of alkali and alkaline earth cations. Chemical Reviews 5: 389-446. RICERS W. P. 1958. Physiology of the hatching of eggs of Ascuris ~zinzbr~~o~des.Nature (London) \ , 18 1: 14 1O1411. ROGERS W. P. 1960. The physiology of infective processes of nematode parasites; the stimulus from the animal host. Proceedings of the RoyalSociety Series B 152: 367-386. SAENCER W. 1980. Cyclodextrin inclusion compounds in research and industry. Angewandte Chemie (International Ed~i~on~ 19: 344-362. SROMOVAD. 1986. The formation of ascarosid layer of the egg-shell in Ascuris lumbricoides. Folk Parasitotogica 33: 169-171. TARR G. E. & FAIRBAIRN D. 1973. Ascarosides of the ovaries and eggs of Ascaris htmbricoides (Nematoda). Lipi& 8: 7-l 6. TARR G. E. & SCHNOES H. K. 1973. Structures of ascaroside aglycones. Archives of B~oche~zist~~and Biophysic.~ 158: 288-296. WATTAL C., MALLA N., KHAX I. A. & AGARWAL S. C. 1985. Reversible inhibition of development and hatching of infective eggs of Ascuris lumbricoides var. hominis. Journal of Parasitology 7 1: 5 18. WHARTON D. A. 1986. A Functional BiologyofNematodes. Croom Helm. London.